WO2003097818A1 - A method for producing transgenic animal mammary glands secreting human erythropoietin (epo). - Google Patents

Abstract

A new process is disclosed for producing EPO, said process comprising the steps of a) constructing a fusion gene BLG-EPO comprising: 1) regulatory elements needed for mammary gland specific expression, ie. 5' flanking sequence, parts of exons and introns, in combination with 3' flanking sequence , derived from B. Taurus lactoglobulin (BLG) gene; 2) human whole genomic EPO DNA encoding sequence, said fusion gene can instantaneously express in mammary glands of goat simulated milk secretion; b) micro-injecting said fusion gene into pronucleus of fertilized eggs of goat, thereby obtaining transgenic goat capable of secreting human EPO in milk specifically and at high level.

Description

 Method for producing human erythropoietin using transgenic animal breast

 Technical field

 The present invention relates to the fields of genetic engineering and transgenic animals, and in particular, the present invention relates to a vector that regulates the specific expression of foreign genes (such as human erythropoietin) in animal breast tissue, and specifically expresses foreign genes in animal breast Genetic approach. The invention also relates to a new method for producing human erythropoietin. Background technique

 Erythropoietin (EPO) is a multifunctional glycoprotein, which can stimulate the proliferation and differentiation of red blood cells. It is mainly used in the treatment of renal anemia and is a valuable medical protein. The production method of EPO is mainly through microbial fermentation. However, the current production process cost is relatively high, equipment investment requires more capital, and the environment is also seriously polluted.

 Animal mammary gland tissue has a strong function of synthesizing protein. If transgenic technology can be used to transfer foreign genes, especially genes with important medical value, such as human erythropoietin gene, human growth hormone gene, etc., it is possible A large amount of medicinal protein is produced. The physiological activities of many proteins in the human body are related to post-translational modifications. Proteins expressed through prokaryotes often lack biological activity due to the lack of post-translational modifications. The expression of these proteins in eukaryotic cell culture requires a higher cost, and the expression of foreign proteins in transgenic animals, whose products are close to natural, have high biological activity, high expression and low cost.

 The gene transferred into the animal by microinjection method, the integration and expression of the genome in the animal is random. If the foreign gene product has strong biological activity, the random or ectopic expression of the foreign gene will be May cause disease or even death of transgenic animals. For example, the ectopic expression of erythropoietin can cause hypererythrocyteemia. To date, there have been no reports in the art of producing EPO by a mammary gland (transgenic sheep or bovine mammary glands).

 Therefore, there is an urgent need in the art to develop new low-cost, low-pollution new EPO production methods. Summary of the Invention

 The purpose of the present invention is to provide a method for producing erythropoietin with low cost and low pollution, that is, using the transgenic animal mammary gland to produce human erythropoietin, thereby expressing a large amount of human erythropoietin in the animal mammary gland.

Another object of the present invention is to provide a transgenic animal that expresses a fusion gene specific to a breast tissue, a vector that efficiently expresses a foreign gene, and a breast-specific EPO. In a first aspect of the present invention, a breast-specific expression fusion gene is provided, and the fusion gene includes the following elements in order from 5 'to 3': the 5 'flanking sequence of bovine lactoglobulin, the human erythropoietin genome DNA sequence, and 3 'flanking sequences of bovine lactoglobulin.

 In a preferred example, the fusion gene includes the following elements in order from 5 'to 3': the 5 'flanking sequence of bovine lactoglobulin, the non-coding sequence of exon 1, the human erythropoietin genomic DNA sequence, and the bovine lactoglobulin Exon 1 coding sequence, bovine globulin intron 1, bovine globulin exon 2, bovine globulin intron 2, bovine globulin exon 3, bovine globulin exon 6, bovine lactoglobulin Intron 6, bovine lactoglobulin exon 7 and bovine lactoglobulin 3 'flanking sequences.

 In a preferred example, the fusion gene further has a linker sequence between the 5 ′ flanking sequence of bovine lactoglobulin and the non-coding sequence of exon 1 and the human erythropoietin genomic DNA sequence; and / or There is also a linker sequence between the erythropoietin genomic DNA sequence and the bovine lactoglobulin exon 1 coding sequence.

 In another preferred example, the 5 'flanking sequence and the exon 1 non-coding sequence of bovine lactoglobulin in the fusion gene have the nucleotide sequence shown in SEQ ID NO: 1;

 The human erythropoietin genomic DNA sequence has the nucleotide sequence shown in SEQ ID NO: 2; bovine lactoglobulin exon 1, bovine lactoglobulin intron 1, bovine lactoglobulin exon 2, and bovine lactoglobulin Intron 2, bovine lactoglobulin exon 3 has the nucleotide sequence shown in SEQ ID NO: 3;

 The bovine lactoglobulin exon 6, the bovine globulin intron 6, the bovine globulin exon 7, and the bovine globulin 3 'flanking sequence have the nucleotide sequence shown in SEQ ID NO: 4.

 In a second aspect, the present invention provides a method for producing a transgenic animal, which includes the steps:

(a) the above-mentioned breast-specific expression fusion gene of the present invention is microinjected into an animal's fertilized egg pronucleus to obtain a fertilized egg in which the fusion gene is integrated in the genome;

 (b) The fertilized egg with the fusion gene integrated in the genome in step (a) is developed into a transgenic animal, and the animal has a specific expression of erythropoietin in its breast.

 Preferably, the animal is selected from the group consisting of sheep and cattle.

 In a third aspect, the present invention provides a method for producing human erythropoietin, comprising the steps of:

(3) The transgenic animal prepared by the method of the present invention is cultured, and the expressed human erythropoietin is isolated and purified from the milk secreted by the animal's mammary gland.

 In a fourth aspect, the present invention provides a vector for regulating specific expression of an exogenous gene in an animal's mammary gland, the vector containing the fusion gene according to claim 1.

 In a fifth aspect, the present invention provides a milk which contains human erythropoietin at a concentration of 100-100,000 OU / ml. More preferably, the milk secreted by the transgenic animals contains human erythropoietin at a concentration of 500 to 50,000 U / ml, more preferably 1,000 to 50,000 U / ml, and most preferably 5000 to 50,000 U / ml.

In a sixth aspect, the present invention provides a transgenic non-human mammal. The milk secreted by the animal in the mammary gland contains 100 to 100,000 U / ml human erythropoietin. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the BLG-EPO fusion gene element.

 Figure 2 is a flowchart of BLG-EPO fusion gene construction.

 Figure 3 is a restriction map of the BLG-EPO fusion gene. The number below each digestion site is the relative position of each site.

 Figure 4 is the integrated detection of the fusion gene BLG-EPO sheep genome. Among them, P is a positive control, N is a negative control, and 73, 132, 134, and 138 are sheep numbers that are positive for integration testing.

 Figure 5 shows the results of detection of erythropoietin mammary gland expression in transgenic sheep. Figure 5A shows the whole protein Ponceau staining after the protein is transferred to the nitrocellulose membrane; Figure 5B is the color development result after antibody hybridization on the cellulose acetate membrane. The lanes are as follows: lane 1 is the molecular weight marker; lane 2 is the EPO standard (Shenyang Sansheng), the molecular weight is 34KD, and the sample load is 30 IU; lanes 3, 4, and 5 are the 134th sheep after lactation, respectively On the 1st day, the goat milk sample was loaded with 0.5ul; lanes 6, 7, and 8 were sheep 138 on the 3rd, 2nd, and 1st day after lactation, and the load was 0.5ul.

 Figure 6 is a standard curve for the detection of EPO activity by the MTT method.

 Figure 7 shows the erythropoietin amino acid sequence expressed by the breast. Italics indicate signal peptide sequences and boldface indicate mature peptide sequences. detailed description

 The present invention first discovered that the 5 'flanking sequence of bovine lactoglobulin and the 3' flanking sequence of bovine lactoglobulin can effectively make foreign genes be specifically expressed in the mammary glands of transgenic animals. The present invention has been completed on this basis.

 In a preferred example of the present invention, the sequence of bovine lactoglobulin used includes: from 5 'to 3', the following elements are included in sequence: the 5 'flanking sequence of bovine globulin, non-coding sequence of exon 1 and Exon 1 coding sequence, bovine globulin intron 1, bovine globulin exon 2, bovine globulin intron 2, bovine lactoglobulin exon 3, bovine globulin exon 6, bovine lactoglobulin Intron 6, bovine lactoglobulin exon 7 and bovine lactoglobulin 3 'flanking sequences. Among them, a more preferred way is to insert a foreign gene between the non-coding sequence of exon 1 and the coding sequence of exon 1 of bovine lactoglobulin.

 Of course, a linker sequence may also be included between the exogenous gene (such as the EPO gene) and the bovine lactoglobulin gene, for example, the 5 'flanking sequence of bovine globulin and the non-coding sequence of exon 1 and the human erythropoietin genomic DNA sequence There is also a linker sequence A between them; and / or a linker sequence B between the human erythropoietin genomic DNA sequence and the bovine lactoglobulin exon 1 coding sequence. The purpose of the adaptor sequence is to facilitate cloning operations. Designing and selecting a variety of linker sequences is within the skill of those skilled in the art. In a preferred example of the present invention, the linker sequence shown is: linker sequence A is 5'-ctcgagtcacc-3 '(SEQ ID NO: 15); the linker sequence B is 5'-ccatgtcgagt-3' (SEQ ID NO: 16).

The invention also provides a vector for regulating the specific expression of foreign genes in animal breast tissue. For The foreign gene (human erythropoietin) is specifically expressed in the mammary gland of the animal. In the present invention, a mammary gland-specific expression vector is constructed using a mammary gland-specific expression control element of the bovine milk protein gene. A preferred vector includes the 5 'flanking sequence of the bovine lactoglobulin gene, partial exon and intron sequences, and the 3' flanking sequence. A multiple cloning site for foreign gene insertion, downstream of the 5 'flanking sequence, and a pGEM-Teasy sequence for replication of the vector in E. coli with the foreign gene inserted thereon.

 In one embodiment of the present invention, the constructed vector has the following sequence elements:

 1.Includes the 5 'regulatory sequence (-1216 ~ + 45 bp) of the bovine lactoglobulin gene (BLG);

 2.Contains bovine lactoglobulin gene exons 1 to exon 3 (+ 48 ~ + 1878 bp), exons 6 to exon 7 (+ 4109 ~ + 4723) and 3 'flanking sequence (734 bp ;).

 In another embodiment of the present invention, a fusion gene BLG-EPO expressing human erythropoietin is constructed based on the specific expression vector. The fusion gene is characterized by: EPO full-length coding sequence genomic DNA 5 ' The BstEII end is linked to the 5 ′ regulatory sequence of BLG and downstream of the non-coding sequence of exon 1, and the 3 ′ end is linked to the upstream of BLG exon 1 coding sequence to the 3 ′ flanking sequence by Ncol. The EPO genomic DNA of the fusion gene is located downstream of the transcription initiation site and is immediately adjacent to the 5'-UTR of the BLG. Therefore, EPO can be transcribed under the action of the BLG promoter. The transcript is a fusion mRNA, and its coding sequence is EPO genomic DNA. The 5'-UTR and 3'-UTR are derived from BLG and EPO. Because EPO genomic DNA provides a stop code, only the EPO protein is encoded, and BLG is not translated.

 The control sequence contained in the vector / fusion gene of the present invention contains a breast-specific expression element, which can ensure that the foreign gene is specifically expressed in the breast.

 In another embodiment of the present invention, when a fusion gene BLG-EPO capable of expressing human erythropoietin specifically in the mammary gland is constructed with this vector and transferred to an animal, human erythropoietin is expressed in a large amount in the animal's mammary gland.

 The transgenic animal that can be used in the present invention is not particularly limited, and may be any mammal. Preferably, the animal is selected from the group consisting of sheep, cattle, and rabbits, and more preferably selected from the group consisting of sheep and cattle. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing conditions Conditions recommended by the manufacturer. Example 1 Construction of EPO breast expression vector

 The gene construction process of this embodiment is shown in Figure 2. The gene construct is the BLG-EPO fusion gene, in which the regulatory sequence is derived from bovine BLG and EPO, and the coding sequence is derived from EPO.

Preparation of pBLG-EPO Source of each component

 1) Bovine BLG5 'flanking sequence and exon 1 non-coding sequence portion

 The following primers were designed and synthesized based on the sequences reported by GENBANK X14710: 5'- cggccgggggtctgctcc -3 '(SEQ ID NO: 5) and 5'- ctcgagggctgcagctggggtcac -3' (SEQ ID NO: 6), using bovine genomic DNA as a template, using PCR method (94 ° C, 5min; 94 ° C, 1min; 60 ° C45s; 72 ° C 90s, 30 cycles) amplified BLG5 'flanking sequence and exon 1 non-coding part sequence (SEQ ID NO: 1) . The PCR product was cloned into pGEM-Teasy vector (Promega) to obtain plasmid pBLG5 '.

 2) Bovine BLG exons 1-3 and exons 6-7 and 3 'flanking sequences

 Based on the sequence reported by GENBANK X14710, the following primers were designed and synthesized: 5'- ctcgagtagtgcctcctgcttgccctg-3 '(SEQ ID NO: 7) and 5'-atcgatcttgaacaccgcagg- 3' (SEQ ID NO: 8), using bovine genomic DNA as a template, PCR (94 ° C, 5min; 94 ° C, 1min; 58 ° C 45s; 72 ° C 90s, 30 cycles) to amplify the exon 1-3 sequence (SEQ ID NO: 3) and PCR amplification The product was cloned into pGEM-Teasy vector to obtain plasmid pBLG 1-3.

 The following primers were designed and synthesized based on the sequence reported by GENBANK X14710: 5'—atcgat gtgagcccctgccggcgc-3 '(SEQ ID NO: 9) and 5'-gcatgccaggcctttctgtc-3 (SEQ ID NO: 10), using bovine genomic DNA as a template , PCR (94 ° C, 5min; 94 ° C, 60s; 56 ° C 60s; 72 ° C 90s, 35 cycles) was used to amplify the BLG exon 6-7 and 3 'flanking sequences (SEQ ID NO: 4), and the PCR amplification product was cloned into pGEM-Teasy vector to obtain plasmid pBLG 6-3 '.

 3) Using human genomic DNA as a template, the following primers were used: 5'-atgagggcccccggtgtg-3 '(SEQ ID NO: 11) and 5'-agtgtccatgggacaggctg-3' (SEQ ID NO: 12), and the full length of EPO was obtained by PCR. Coding sequence genomic DNA and part of 5'-UTR! ] 3'-UTR sequence (SEQ ID NO: 2), and cloned it into the pGEM-Teasy vector to obtain the plasmid pEPO.

2. Construction of pBLG-EPO

 CBL I and Sal I double digested pBLG6-3 'to recover the exon 6-3' fragment (about 1350bp). This fragment was ligated to the Cla I and Sal I sites of pBLGl-3 to obtain pBLGl-3 '; Xho I and Sal I double digestion of pBLGl-3 'to recover BLG1-3' fragment (approximately 3.1 Kb). This fragment was connected to the Xhol and Sal I sites of pBLG5 'to obtain pBLG; BstE II and Nco I were double digested to recover pEPO. An EPO fragment (approximately 2.3 KB) was blunt-ended to the Xho l site of pBLG to obtain pBLG-EPO. (See Figure 2 for the construction process).

 For digestion map of pBLG-EPO breast-specific fusion gene, see Figure 3.

 In pBLG-EPO, the EPO-BLG5 'linker and its adjacent sequences are as follows:

Transcription start site BLG5 '-UTR linker sequence EP0 5' -UTR

CCTCCACTCCCTGCAGAGCTCAGAAGCGTGACCCCAGCTGCAGCCCTC GCTGAGGGACCCCGGCCAGGCGCGGAGATG (SEQ ID NO: 13)

Start codon The EPO and BLG 3 'linker and adjacent sequences are as follows:

EPO 3 * -UTR connector sequence BLG3 '-UTR

C 丌 GTGGCGC (SEQ ID NO: 14) See FIG. 1. In the human erythropoietin mammary gland specific expression vector pBLG-EPO or the fusion gene BLG-EPO, the functions of each element are as follows:

 The shaded box is the 5 'flanking sequence of BLG. This part is the BLG promoter, which is responsible for driving the expression of foreign genes and ensuring that the expression of foreign genes is breast-specific. One represents the BLG5 'untranslated region (5'-UTR), and 5'-UTR provides a ribosome binding site for a foreign gene translation-encoding protein. The detailed sequence is shown in Figure 4 and SEQ ID NO: 1.

 The open box is the full-length coding sequence of the foreign gene EPO. Include translation initiation and termination codons. See SEQ ID NO: 2 for the sequence details.

 The solid boxes are bovine BLG exon 1 to exon 3, exons 6 to 7 and 3 'flanking sequences, which serve to provide transcription termination signals and add Poly A signals. The exon part provides a 3 'untranslated region to the exogenous gene, and the intron part and the 3' flanking sequence are related to the regulation of breast-specific high-efficiency expression. For the sequence details, see SEQ ID NO: 3, and SEQ ID NO: 4.

 The amino acid sequence of human EPO is shown in Figure 7 and SEQ ID NO: 17. Example 2 Preparation of fusion gene pBLG-EPO transgenic goat

 1. Preparation of transgenic plasmid DNA

 1) The plasmid pBLG-EPO is used to transform E. coli DH52, and the DH52 strain containing the pBLG-EPO plasmid is cultured in liquid. The culture amount can be 100ml-1000ml according to the needs. Then, the bacteria are collected, and the pBLG- EPO (For detailed operations, refer to the Handbook of Molecular Cloning, edited by Manny Antis).

 The extracted pBLG-EPO plasmid was digested with Notl, agarose gel electrophoresis, and Sephaglass

The Babndprep (pharmacia biotech) kit was used to recover and purify the 6.82Kb fragment.

 2) Dilute the purified DNA to 2ug / ml with a microinjected DNA dilution solution, centrifuge (12000rpm, 30min), and aliquot it for microinjection.

 2. Preparation of transgenic animals

 The fusion gene BLG-EPO was introduced into the male pronucleus of a fertilized egg of a dairy goat by microinjection, and then transplanted into the oviduct of a recipient goat that was in estrus synchronously. Example 3. Detection of integration of fused gene pBLG-EPO transgenic goat genome

Sample processing For non-transgenic goats and transgenic goats to be tested, sheep ears (less than 1 cm in length) were obtained according to conventional procedures and stored at -20 ° C for future use.

 Detection method

 1) Rapid extraction of genomic DNA: Put sheep ears in a 1.5ml centrifuge tube, add 0.5ml Lysis Buffer (4M Urea power, 10mM EDTA (pH8.0), 0.5% Sarkosyl (Sigma L5125), O. IM Tris-Cl (pH 8.0), 0.2M NaCl), 50 μl of proteinase K (10 mg / ml) was shaken in a 55 ° F. hybridization oven overnight. Centrifuge (14000 rpm) for 5 min, transfer the supernatant to a clean centrifuge tube, add 1 ml of absolute ethanol, mix gently, shake vigorously, use a Tip to gently lift the flocculent DNA pellet, and use 250-300 μ 1 of 0.1 Resuspend DNA in X TE or double distilled water, mix by pipetting, and store at 4 ° C.

 2) Digestion and electrophoresis: take about 10 micrograms of genomic DNA and use restriction enzyme EcoR l at 37

Digest at ° C for more than 12 hours. The digested product was electrophoresed on a 1% agarose gel with a voltage of 1-2 V / cm for more than 12 hours. _

 Denaturation and neutralization: Add denaturing solution (1.5M NaCl 0.5M NaOH) to denature the gel for 45 minutes (lightly shake), add neutralizing solution (1.5M NaCl 0.5M Tris-Hcl (PH 8.0) and shake gently for 45 minutes ( Shake slightly)

 3) Transfer film: Spread the nylon film evenly on the glue, spread 3 sheets of 3MM Waterman absorbent paper on it, then spread the paper tower (5-10 cm), press the weight, transfer the film for 18 hours; rinse the film with 5 X SSC To remove gel fragments on the surface of the membrane; dry at room temperature and fix at 80 ° C for 2 hours.

 4) Probe preparation: The plasmid pBLG-EPO was double digested with EcoR l and Xho l, and a 3.5 kb band was recovered. The yield should be between 500 ng and 1000 ng. The probe was labeled with a random primer labeling kit.

 5) Pre-hybridization and hybridization: transfer membrane in pre-hybridization solution (5 X SSPE, 5 X Denhardfs Solution,

0.1% SDS, 100 μg / ml nonhomologous Salmon sperm blocking DNA, 50% Formamide) at 42 ° C for 4 hours. The probe was added to the pre-hybridization solution and hybridized at 42 ° C for 16 hours.

 6) Film washing and tablet washing: Wash Solution (1 X SSC, 0.1% SDS) at 65 ° C for 1 hour. Repeat this step until the background drops below 10. Fix the washed film with safety film, put X-ray film in the dark room, and store at -70Ό for two days. Remove the X-ray film in the dark room, develop it for 5 minutes, fix it for 15 minutes, check the result displayed on the light film,

 Test results

 The results are shown in Figure 4. Lane P is a positive control: the transgenic component BLG-EPO is digested with EcoR1; lane N is a negative control: untransgenic goat genomic DNA; the remaining lanes are the genomic DNA of the transgenic goat to be tested. The results showed that BLG-EPO was integrated in the genomic DNA of transgenic goats (73, 132, 134, 138). Example 4. Western blot to detect human erythropoietin in transgenic goat milk

Sample processing 1) Artificial milking of transgenic milk goats:

 Milk goats were selected and intramuscularly injected with estradiol benzoate and progesterone twice a day at 2 mg and 20 mg each for seven days. Then, intramuscularly, 0.5 mg reserpine was injected on days 8, 10, 12, and 14, respectively. Milk was collected for EPO expression detection on day 12

 Collect goat milk, degrease (4000rpm, 4 ° C, 20min), add equal volume of TBS buffer

(O.lmol / L Tris-base, 0.3mol / L NaCl, 0.2mol / L EDTA, PH6.5), sterilize through a 0.22um filter, add 1 X SDS gel loading buffer to the sample, Boil for 3 minutes at ° C to denature the protein.

 Detection method

 Protein electrophoresis: SDS-PAGE gel electrophoresis of the processed samples, laminated gel: 4% gel concentration, 50V; separation gel: 7.5% gel concentration, 100V.

Protein transfer: The protein transfer was performed at 80 mA for 2 hours after electrophoresis. The protein was transferred to a nitrocellulose membrane, and the transfer effect was detected by Ponceau staining. The transfer membrane was washed with Tris-NaC solution (0.05m _O l / L Tris-base, 0.15mol / L NaCl, pH7.5) to remove the Ponceau red dye bound to the protein, and then used to hybridize the antibody.

 Antibody hybridization: Put the transfer membrane into a plate, add an appropriate amount of blocking solution (10% skimmed milk powder), add the first antibody (rat anti-human erythropoietin antibody) at a concentration of 1: 3000, and place it on a gentle shaker at room temperature Hybridize for four hours. After the hybridization, rinse the filter 3 times with Tris-NaCl solution for 10 minutes each time. Add enzyme-linked secondary antibody at a concentration of 1: 2000 and hybridize for two hours at room temperature. After hybridization, rinse with Tris-NaCl solution The membrane was applied three times for 10 minutes each, and then a color reaction was performed.

 Color development: Add an appropriate amount of substrate (BCIP / NBT for alkaline phosphatase) to the membrane, and react for about 20 minutes in the dark to see the protein bands.

 3. Test results

 The results are shown in Figure 5, where lane 1 is the molecular weight standard; lane 2 is the positive control EPO standard (Shenyang Sansheng):; lanes 3, 4, 5, 6, 7, and 8 are the transgenic goat milk protein to be tested.

 The results showed that specific bands of erythropoietin were shown in the milk of transgenic goats 134, 138 (lanes 3, 4, 5, 6, 7, 8). Example 5. In vitro biological activity of human erythropoietin (EPO) in transgenic goat milk (MTT method)

 Vigorous energy metabolism in living cells, especially proliferating cells. The use of succinate dehydrogenase produced during mitochondrial energy metabolism can reduce the pale yellow MTT to blue-violet crystals deposited around the nucleus. Directly proportional. Using the growth-dependent properties of 32D cells for EPO, the EPO activity in the culture medium can be detected.

 1. Experimental materials:

1640 culture solution: Prepare according to the instructions and save at 4 degrees. Basal culture medium: 1640 medium is supplemented with 10% calf serum, 1 * P / S, and stored at 4 degrees. Complete medium: Add EPO to the base medium to a final concentration of 1-2 U / ml, and store at 4 ° C.

PBS: 8g of NaC1, 0.2g of KC1, 0.24g of Na ₂ HPO ₄ , KH ₂ PO ₄ and distilled water to make 1000 ml. Sterilize at 121 ° C for 15 minutes.

 Thiazole blue MTT solution: Prepare a 5.0mg / ml solution with PBS, filter and sterilize through a 0.22um filter,

Store at 4 degrees away from light.

 Lysate: 10% SDS, 0.01mol / ml HCl.

 32D cell culture: 32D cells were cultured in complete medium for 24-48 hours for EPO activity determination. EPO standard: Shenyang Sansheng Yibiao 3000U / ml, stored at 4 ° C.

 2. Experimental steps:

 Blank goat milk preparation:

 Blank goat milk is sterilized with a needle filter. Dilute to 10% solution with basal medium.

 Sample solution preparation:

 Sterilize and filter. Goat milk samples are sterilized with a needle filter, diluted with a basic culture solution to a 10% solution, and diluted with 10% blank goat milk to different concentration gradients.

 Preparation of standard sample dilution:

 Take Yibiao to prepare a 12.8U / ml solution with 10% blank goat milk, and then dilute with blank goat milk to 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05U / ml.

 Prepare the cell suspension:

 Take a sufficient amount of 32D cell culture to collect 32D cells by centrifugation, wash them with basal culture solution three times, resuspend in the basal culture solution and count to make 400,000 cells per ml for use. In a 96-well plate, add 100ul of the standard series and 100ul of sample, as shown in the table below.

 Add cell suspension and culture:

Add 100ul of cell suspension to each well, incubate at 37 ° C, and 5% CO _{2 for} 44 hours.

 Join MTT and train:

Add 10ulMTT solution, 37 to the enzyme well. C, 5% CO _{2 for} 4 hours

 Add lysate and incubate:

 Add 100ul lysate to the enzyme well and incubate at 37 ° C for 18-24 hours

 Determination of optical density:

 The color is measured on a microplate reader, the measurement wavelength is 590nm (570-600, and the reference wavelength is 630nm is optional), and the measurement results are recorded.

 3. Results

The standard curve for the detection of EPO activity by the MTT method is shown in FIG. 6. Logarithmic logarithm of standard series concentration u / ml OD590 was used for linear regression to calculate the sample EPO concentration. The results showed that BLG-EPO integrated sheep 132, 134, and 138 expressed EPO in breasts of 117 U / ml, 28000 U / ml, and 19038 U / ml milk, respectively.

 Development of MTT method for the determination of EPO activity standard curve

在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单 独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领 域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附 权利要求书所限定的范围。

All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

Rights request  A breast-specific expression fusion gene, characterized in that the fusion gene comprises the following elements in order from 5 'to 3': the 5 'flanking sequence of bovine lactoglobulin, the human erythropoietin genomic DNA sequence, and bovine milk globules Protein 3 'flanking sequence.  2. The fusion gene according to claim 1, wherein the fusion gene comprises the following elements in order from 5 'to 3': 5 'flanking sequence of bovine lactoglobulin, non-coding sequence of exon 1, human erythrocyte Genome DNA sequence, bovine globulin exon 1 coding sequence, bovine globulin intron 1, bovine globulin exon 2, bovine globulin intron 2, bovine globulin exon 3, bovine globules Protein exon 6, bovine lactoglobulin intron 6, bovine lactoglobulin exon 7, and bovine lactoglobulin 3 'flanking sequences.  The fusion gene according to claim 1, further comprising a linker sequence between the 5 'flanking sequence of bovine lactoglobulin and the non-coding sequence of exon 1 and the human erythropoietin genomic DNA sequence; and There is also a linker sequence between the human erythropoietin genome DNA sequence and the bovine lactoglobulin exon 1 coding sequence.  The fusion gene according to claim 1, wherein the 5 'flanking sequence of bovine lactoglobulin and the non-coding sequence of exon 1 have the nucleotide sequence shown in SEQ ID NO: 1;  The human erythropoietin genomic DNA sequence has the nucleotide sequence shown in SEQ ID NO: 2; bovine lactoglobulin exon 1, bovine lactoglobulin intron 1, bovine lactoglobulin exon 2, and bovine lactoglobulin Intron 2, bovine lactoglobulin exon 3 has the nucleotide sequence shown in SEQ ID NO: 3;  The bovine lactoglobulin exon 6, the bovine globulin intron 6, the bovine globulin exon 7, and the bovine globulin 3 'flanking sequence have the nucleotide sequence shown in SEQ ID NO: 4.  5.—A method for producing a transgenic animal, characterized in that it includes steps:  (1) The breast-specific expression fusion gene according to claim 1 is microinjected into an animal's fertilized egg pronucleus to obtain a fertilized egg in which the fusion gene is integrated in the genome;  (2) The fertilized egg in which the fusion gene is integrated in the step (1) is developed into a transgenic animal, and the animal has a specific expression of erythropoietin in its breast.  The method of claim 5, wherein the animal is selected from the group consisting of sheep and cattle.  7. A method for producing human erythropoietin, characterized in that it includes steps:  (3) culturing the transgenic animal prepared by the method according to claim 5, and isolating and expressing the expressed human erythropoietin from milk secreted from the mammary glands of the animal.  8. A vector for regulating the specific expression of a foreign gene in an animal's mammary gland, characterized in that the vector contains the fusion gene according to claim 1.  9. A milk, characterized in that it contains human erythropoietin in a concentration of 100-100,000U / ml.  10. A transgenic non-human mammal, characterized in that the milk secreted by the animal in the mammary gland contains 100-100,000 U / ml human erythropoietin.